Methods and materials for detecting mutations in quasispecies having length polymorphisms

ABSTRACT

The present invention is directed to a method for detecting the presence or absence of a mutation of interest in the nucleic acid of a pathogen, wherein the mutation of interest is located adjacent to a length polymorphism defining multiple quasispecies of the pathogen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/603,195, filed Aug. 20, 2004, and U.S. Provisional Application No.60/603,337, filed Aug. 20, 2004.

BACKGROUND OF THE INVENTION

The present invention generally relates to methods and materials fordetecting the presence or absence of a mutation of interest in apathogen. The present invention also relates to particular methods andprimers for determining the presence or absence of a mutation amongmultiple human immunodeficiency virus (HIV-1) quasispecies present in asample from a single patient.

The nucleic acid sequence of pathogens are often subject to a highmutation rate, giving rise to a variety of polymorphic variants. Forexample, human immunodeficiency virus, a member of the Lentivirus groupof retroviruses, and the primary causative agent of Acquired ImmuneDeficiency Syndrome (AIDS), or AIDS-related complex (ARC), typicallyundergoes frequent mutation. The HIV-1 RNA genome comprises variousgenes that encode proteins necessary for the replication of HIV-1. Likeall other retroviruses, it has an RNA genome which is replicated bymeans of the viral reverse transcriptase (RT) enzyme, which copies thesingle-stranded viral RNA genome into a double-stranded DNA/RNA hybrid,resulting in integration of the DNA provirus into the host cell genome.The RT enzyme lacks a 3′exonuclease activity which normally helps the“proof-reading” function of a polymerase enzyme to repair errors.Consequently, the RT enzyme makes at least one error during everytranscription of 10,000 bases copied, resulting in errors that areresponsible for the high mutation rate of HIV-1.

The HIV-1 RNA genome also includes a gene that encodes the envelopeglycoprotein (env), which consists of two principle subunits—the gp120surface glycoprotein and the gp41 transmembrane glycoprotein. The gp41subunit encodes transmembrane proteins that facilitate fusion of theHIV-1 virus to the outer cell membrane of CD4 cells. Because the HIV-1env protein plays a critical role in the initial infection of CD4 cells,it has been a primary target in the search for drugs that can inhibitthe interaction of proteins responsible for fusion of HIV-1 to cells,thereby inhibiting HIV infection. One particular target in the gp41subunit of the HIV-1 env protein is the heptad repeat 1 (HR1) and heptadrepeat 2 (HR2) domains, which have been shown to play a key role infacilitating the conformational changes required for fusion of viral andcellular membranes. Because many anti-retroviral drugs target the HIV-1env protein in order to inhibit entry of HIV-1 into the cell, manymutations responsible for HIV-1 drug resistance arise in this region.

Various drugs that are presently available to treat HIV fall into threedifferent classes—nucleoside reverse transcriptase inhibitors (NRTIs),non-nucleoside reverse transcriptase inhibitors (NNRTIs), and proteaseinhibitors (PIs). Presently available anti-retroviral compounds used totreat AIDS suffer from certain disadvantages, including transient CD4cell count effects, incomplete inhibition of viral replication, toxicityat prescribing doses, and emergence of resistant forms of the virus.Even with the advent of combination therapies, many patients remainunable to achieve or maintain complete viral suppression even withanti-retroviral compounds. As a result of incomplete viral suppression,coupled with the very high mutagenicity rate of HIV virus (due to theerror-prone nature of the viral RT enzyme) and the genetic variabilityof the virus, many HIV variants with decreased drug susceptibility havearisen. For example, the use of Enfuvirtide (Enf, previously referred toas T-20), the first of a new class of anti-HIV drugs that inhibit fusionof HIV with a host cell, has resulted in the emergence of resistancemutations in the first heptad repeat domain of gp41 (HR1) that have beenlinked to T-20 treatment failure.

By identifying mutations associated with HIV-1 drug resistance tospecific anti-retroviral drugs before therapeutic intervention, theparticular course of therapeutic intervention can be optimized byselecting and administering drugs to which the virus is mostsusceptible. Mutations can be detected by various techniques, the mostdirect and reliable of which is sequencing of the viral DNA(genotyping). In the case of clinical genotyping, where critical andeven life-saving decisions relating to therapeutic intervention are madebased on the genotyping results, confirmation of sequencing results bycomparison with the sequence of the complementary strand of DNA is evenmore critical. The effectiveness of genotyping, and the ability toobtain bidirectional confirmation of sequence results is, however,compromised when multiple species of a pathogenic vector are present ina single patient sample. Because a patient sample containing mixedspecies of a pathogenic vector will contain multiple variants of the DNAsequence, sequencing will show multiple bases at a particular locationand, in the case of insertion or deletion mutations, will show multiplebases at each location over an entire region of the DNA as a result of ashift in the reading frame, thus confounding the results and precludingcomplementary strand confirmation of the sequence and identification ofclinically relevant mutations within that sequence. Pathogenic vectorsthat are present in the form of multiple species within a patient samplehave therefore become increasingly refractory to clinical genotypingefforts, and have become a significant challenge to creating diagnosticassays to detect clinically relevant mutations, such as mutations thatcause viral resistance to particular therapeutic drugs.

Consequently, there is a need to develop more accurate and reliablegenotyping methods that are amenable to complementary strandconfirmation in clinical settings, and that are capable of detecting andidentifying clinically relevant mutations in a pathogenic vector presentin the form of multiple quasispecies within a patient sample.

SUMMARY OF THE INVENTION

The present invention provides improved methods and materials forclinical genotyping of a pathogen, such as HIV, present in a patientsample containing multiple quasispecies of the pathogen.

In a particular aspect, the present invention relates to methods andmaterials for detecting the presence or absence of a mutation ofinterest in a pathogen present in a sample containing multiplequasispecies of the pathogen having mixed length polymorphisms, whereinthe mutation of interest is located adjacent to the length polymorphism

In a particular aspect, the present invention is directed to methods andmaterials for detecting the presence or absence of a mutation ofinterest in a patient sample containing mixed quasispecies of apathogen, wherein the mutation of interest is located adjacent to apredetermined length polymorphism, such as an insertion mutation or adeletion mutation, which results in quasispecies of different nucleicacid sequence lengths. In a particular aspect, the present inventionprovides methods and primers for improved accuracy in genotyping anHIV-1 virus having length polymorphisms, which may be present in apatient sample containing mixed quasispecies. The improved methods andmaterials of the present invention may improve therapeutic interventionand treatment of infectious diseases, including, for example, AIDS.

The methods and primers of the present invention were developed as aresult of the initial discovery that amplification and sequencing of theentire HIV-1 env gene fails to provide reliable complementary strandconfirmatory data necessary to identify and confirm the existence ofimportant drug resistance associated mutations, identification of whichis essential to therapeutic intervention. The gp41 region of HIV-1 envgene contains a first heptad repeat domain (HR1) and a second heptadrepeat domain (HR2). The region surrounding the first heptad repeatdomain (HR1) is subject to frequent insertion or deletion mutations,resulting in mixed HIV-1 populations having drug resistance mutationswithin the HR1 domain, but also containing multiple quasispecies havinglength polymorphisms. The presence of mixed length polymorphisms amongquasispecies in a single patient sample confounds efforts to obtainconfirmatory sequence of the complementary strand with primers coveringa larger region, because the mixed length polymorphism mutation thatoccurs between one of the outer primers and the mutations of interestresults in complementary primer extension products having sequences ofdifferent lengths in the region between the primers, which in turnresults in overlayed and out of frame sequence signals being generated.The method of the present invention improves reliability of genotypingby sequencing the region of the mutation of interest, exclusive of theregion containing the length polymorphism. The primer set may be usedalone or in conjunction with a secondary primer set that includes theregions of variability to confirm genotypes for samples that cannot bereliably characterized using other sequencing primers.

While the methods of the present invention were developed initially withrespect to the HIV-1 gene, such methods are nevertheless applicable toany pathogen having length polymorphisms among multiple quasispecies.Accordingly, the present invention is directed to a method for detectingthe presence or absence of a mutation of interest in the nucleic acid ofa pathogen, such as HIV-1, wherein the mutation of interest is locatedadjacent to a length polymorphism defining multiple quasispecies of thepathogen, comprising:

-   -   a) obtaining from the patient sample a double-stranded DNA        template encompassing the mutation of interest;    -   b) sequencing a first strand of a region of the DNA template        containing the mutation of interest, exclusive of the length        polymorphism;    -   c) sequencing a second strand of a region of the DNA template        containing the mutation of interest, exclusive of the length        polymorphism; and    -   d) comparing the sequence of the first strand with the sequence        of the second strand to obtain complementary strand confirmation        of the sequence of the mutation of interest.

In another aspect, the present invention is directed to a method fordetecting the presence or absence of a mutation of interest in thenucleic acid of a pathogen, such as HIV-1, wherein the mutation ofinterest is located adjacent to a length polymorphism defining multiplequasispecies of the pathogen, comprising:

-   -   a) obtaining from the patient sample a double-stranded DNA        template encompassing the mutation of interest;    -   b) amplifying a first region of the DNA template containing both        the mutation of interest and the length polymorphism;    -   c) sequencing a first strand of a region of the DNA template        containing the mutation of interest, exclusive of the length        polymorphism;    -   d) sequencing a second strand of a region of the DNA template        containing the mutation of interest, exclusive of the length        polymorphism; and    -   e) comparing the sequence of the first strand with the sequence        of the second strand to obtain complementary strand confirmation        of the sequence of the mutation of interest.

In yet another aspect, the present invention is directed to a method fordetecting the presence or absence of a mutation of interest in thenucleic acid of a pathogen, such as HIV-1, wherein the mutation ofinterest is located adjacent to a length polymorphism defining multiplequasispecies of the pathogen, comprising:

-   -   a) obtaining from the patient sample a double-stranded DNA        template encompassing the mutation of interest;    -   b) amplifying a first genetic region of the DNA template        comprising the mutation of interest, exclusive of the length        polymorphism;    -   c) sequencing a first strand of a region of the DNA template        containing the mutation of interest, exclusive of the length        polymorphism;    -   d) sequencing a second strand of a region of the DNA template        containing the mutation of interest, exclusive of the length        polymorphism; and    -   e) comparing the sequence of the first strand with the sequence        of the second strand to obtain complementary strand confirmation        of the sequence of the mutation of interest.

In a one aspect of the invention, the mutation of interest is located ina region of HIV selected from the group consisting of: gp41 and gp120.In another aspect of the invention, the mutation of interest is locatedin the HIV-1 envelope glycoprotein gp41. In yet another aspect of theinvention, the mutation of interest is located within positions 36-45gp41.

In another aspect, the present invention is directed to anoligonucleotide primer capable of generating primer extension productscorresponding to the region consisting essentially of nucleotides7329-7614 of SEQ ID NO:1, 7388-7549 of SEQ ID NO:1 or 7492-7522 of SEQID NO:1, fragments thereof of 15 or more nucleotides.

In another aspect, the present invention is directed to a combination offorward and reverse primers, selected from the group consisting of SEQID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 andfragments thereof of 15 or more nucleotides.

In one aspect, the present invention is directed to a method and primersfor genotyping the gp41 subunit of the HIV-1 env protein, whichcomprises determining the sequence of a region encompassing both the HR1and HR2 domains of the gp41 subunit of the HIV-1 env protein.

In another aspect, the present invention includes primers selected fromthe group consisting of one or more of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, and fragments thereof of 15 or more nucleotides.

In another aspect, the present invention is directed to a primercombination comprising a set of bi-directional sequencing primersencompassing a region encompassing the HR1 and HR2 domains of HIV-1,wherein the primer combination comprises:

-   -   (a) a forward primer selected from the group consisting of one        or more of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,        SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID        NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,        and fragments thereof of 15 or more nucleotides; and    -   (b) a reverse primer selected from the group consisting of one        or more of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID        NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,        SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID        NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and fragments        thereof of 15 or more nucleotides.

The selected primers, one or more from each group, can be used asreverse transcription, amplification and sequencing primers.

The primers are packaged in a suitable genotyping kit. Such a kit mayinclude reagents in addition to the primers, such as an RNase inhibitor,a reverse transcriptase, a polymerase, and/or DNTP and ddNTP feedstocks.In a preferred aspect, the present invention is directed to a kit fordetecting the presence or absence of a mutation of interest in thenucleic acid of a pathogen, wherein the mutation of interest is locatedadjacent to a length polymorphism defining multiple quasispecies of thepathogen, comprising a first primer for sequencing a first strand of aregion of a DNA template containing the mutation of interest and asecond primer for sequencing a second strand of a region of the DNAtemplate containing the mutation of interest, wherein the region definedby the first primer and second primer excludes the length polymorphism.

The primers are employed in the method of the invention, as appropriate.In accordance with this method, a sample suspected of containing theHIV-1 virus is treated to recover viral RNA. The recovered viral RNA isreverse transcribed to DNA, which is sequenced using the primers of theinvention. The resulting sequence information is used to establish thegenotype of the tested virus, i.e., to determine to which subtype,species or quasispecies the virus in the sample belongs, or to determinethe presence or absence of the mutation of interest. The method of theinvention may be practiced in parallel with genotyping procedures thatare designed to evaluate multiple viral species. Alternatively, themethod of the invention is practiced on samples that have previouslybeen the subject of a failed attempt to obtain reliable sequenceinformation for the purpose of genotyping the infectious pathogen.

DETAILED DESCRIPTION OF THE INVENTION

While the terminology used in this application is standard within theart, the following definitions of certain terms are provided to assureclarity.

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation. Numeric ranges recited herein are inclusive of thenumbers defining the range and include and are supportive of eachinteger within the defined range. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUBMB Nomenclature Commission.Nucleotides, likewise, may be referred to by their commonly acceptedsingle-letter codes. Unless otherwise noted, the terms “a” or “an” areto be construed as meaning “at least one of.” The section headings usedherein are for organizational purposes only and are not to be construedas limiting the subject matter described. All documents, or portions ofdocuments, cited in this application, including but not limited topatents, patent applications, articles, books, and treatises, are herebyexpressly incorporated by reference in their entirety for any purpose.In the case of any amino acid or nucleic sequence discrepancy within theapplication, the figures control.

As used herein, the term “pathogen” means an infectious agent orexogenous vector that originates or is initially produced outside of,but is present in a host organism, such as a viral, bacterial, fungal orprotozoan organisms. The method and reagents of the present inventionare advantageously used to genotype a pathogen that is present in thehost organism in the form of multiple quasispecies, wherein thequasispecies are characterized by having one or more alleles of a lengthpolymorphism that give rise to length polymorphisms among thequasispecies.

As used herein, the term “quasispecies” means a species ofself-replicating organism that contains a different genetic sequence asa result of the incorporation or deletion of alternative or additionalgene sequences, either correct or erroneous. Quasispecies may result,for example, from the genetic copy process of other gene sequences thatare already present. Quasispecies typically arise in the context of theevolutionary processes of self-replicating macromolecules such as RNA orDNA of organisms, such as infectious pathogens, including bacteria andviruses. As used herein, the term “quasispecies” includes both thevariant species and the wild-type species from which the variant specieswas derived.

As used herein, the term “length polymorphism” means any mutation in thegenetic sequence that results in a quasispecies having nucleic acidsequence of a different length. Length polymorphism include, but are notlimited to, for example, insertion mutations, deletion mutations, andsubstitution mutations that result in a different nucleic acid sequencelength. In automated sequencing of samples containing multiplequasispecies, the sequencing trace will include data from two differentsequences (i.e., the reference sequence and the sequence containing thelength polymorphism), resulting in multiple peaks being superimposed ata given nucleotide base position. In the context of the presentinvention, the phrase “length polymorphism defining multiplequasispecies of a pathogen” means the genetic locus with respect towhich there exists length polymorphisms that result in multiplequasispecies of the pathogen. Length polymorphisms refer to the variousalleles or species of a pathogen that result in different quasispecies,and include the “wild-type” or “reference” species with respect to whichthe length polymorphism is defined.

As used herein, the term “adjacent” is used in reference to the locationof the mutation of interest relative to a length polymorphism. Amutation of interest is considered to be “adjacent” to a lengthpolymorphism when its location is sufficiently proximate that standardamplification and/or sequencing primers encompasses both the mutation ofinterest and the length polymorphism. While the precise distance betweenthe mutation of interest and the length polymorphism is not critical,any mixed length polymorphism mutation that occurs between one of theouter primers and the mutations of interest causes the sequence qualityto decline due to overlayed and out of frame sequences being generated.The methods and materials of the present invention are advantageouslyemployed when primers are utilized that can exclude any or all lengthpolymorphism(s) that generate such overlayed and out of frame sequences.

As used herein, the term “sample” refers to a biological sample obtainedfrom a patient or group of patients that may contain a nucleic acidanalyte from a pathogen having multiple quasispecies. Patient samplesinclude samples of tissue or fluid isolated from an individual orindividuals, including but not limited to, for example, skin, plasma,serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, bloodcells, organs, tumors, and also to samples of in vitro cell cultureconstituents (including but not limited to conditioned medium resultingfrom the growth of cells in cell culture medium, recombinant cells andcell components).

As used herein, the term “amplification” means the process of increasingthe relative abundance of one or more specific genes or gene fragmentsin a reaction mixture with respect to other genes. Numerousamplification methods are available and known to those in the art. Suchmethods typically utilize the technique of polymerase chain reaction (orPCR) or some other primer extension based methodology. PCR methods aredescribed, for example, in U.S. Pat. Nos. 4,683,194, 4,683,195 and4,683,202, and 4,800,159 which are incorporated herein by reference. Themethod is also explained in texts such as Current Protocols in MolecularBiology, (Eds. Ausubel, F. M. et al., (John Wiley & Sons; 1995)), K.Mullis, Cold Spring Harbor Symp. Quant. Biol., 51:263-273 (1986); and C.R. Newton & A. Graham, Introduction to Biotechniques: PCR, 2.sup.nd Ed.,Springer-Verlag (New York: 1997), the contents of which are incorporatedherein by reference. PCR involves the use of pairs of primers, one foreach complementary strand of the duplex DNA (wherein the coding strandis referred to as the “sense strand” and its complementary strand isreferred to as the “anti-sense strand), that will hybridize at siteslocated on either side of a region of interest in a gene. Chainextension polymerization is then carried out in repetitive cycles toincrease the number of copies of the region of interest exponentially.To briefly summarize, in the first step of the PCR reaction, the nucleicacid molecules of the sample are transiently heated, and then cooled, inorder to denature double stranded molecules. Forward and reverse primersare present in the amplification reaction mixture at an excessconcentration relative to the sample target. When the sample isincubated under conditions conducive to hybridization andpolymerization, the primers hybridize to the complementary strand of thenucleic acid molecule at a position 3′ to the sequence of the regiondesired to be amplified that is the complement of the sequence whoseamplification is desired. Upon hybridization, the 3′ ends of the primersare extended by the polymerase. The extension of the primer results inthe synthesis of a DNA molecule having the exact sequence of thecomplement of the desired nucleic acid sample target. The PCR reactionis capable of exponentially amplifying the desired nucleic acidsequences, with a near doubling of the number of molecules having thedesired sequence in each cycle. Thus, by permitting cycles ofhybridization, polymerization, and denaturation, an exponential increasein the concentration of the desired nucleic acid molecule can beachieved. The amplified polynucleotide may be used as the template for asequencing reaction. Gelfand et al. have described a thermostableenzyme, “Taq polymerase”, derived from the organism Thermus aquaticus,which is useful in this amplification process (see U.S. Pat. Nos.4,889,818; 5,352,600; and 5,079,352 which are incorporated herein byreference). Alternative amplification techniques such as NASBA, 3 SR, QbReplicase, and Branched Chain Amplification are known and available topersons skilled in the art. The term “RT-PCR” refers generally toamplification which includes a reverse transcription step to permitamplification of RNA sequences.

As used herein, the term “sequencing” means the determination of theorder of nucleotides in at least a part of a gene. A well known methodof sequencing is the “chain termination” method first described bySanger et al., PNAS (USA) 74(12): 5463-5467 (1977) and detailed inSequenase® 2.0 product literature (Amersham Life Sciences, Cleveland)and more recently elaborated in European Patent EP-B1-655506, thecontent of which are all incorporated herein by reference. In thisprocess, DNA to be sequenced is isolated, rendered single stranded, andplaced into four vessels. In each vessel are the necessary components toreplicate the DNA strand, which include a template-dependent DNApolymerase, a short primer molecule complementary to the initiation siteof sequencing of the DNA to be sequenced and deoxyribonucleotidetriphosphates for each of the bases A, C, G and T, in a buffer conduciveto hybridization between the primer and the DNA to be sequenced andchain extension of the hybridized primer. In addition, each vesselcontains a small quantity of one type of dideoxynucleotide triphosphate,e.g. dideoxyadenosine triphosphate (“ddA”), dideoxyguanosinetriphosphate (“ddG”), dideoxycytosine triphosphate (“ddC”),dideoxythymidine triphosphate (“ddT”). In each vessel, each piece of theisolated DNA is hybridized with a primer. The primers are then extended,one base at a time to form a new nucleic acid polymer complementary tothe template DNA. When a dideoxynucleotide is incorporated into theextending polymer, the polymer is prevented from further extension.Accordingly, in each vessel, a set of extended polymers of specificlengths are formed which are indicative of the positions of thenucleotide corresponding to the dideoxynucleotide in that vessel. Thesesets of polymers are then evaluated using gel electrophoresis todetermine the sequence.

Sequencing of polynucleotides may be performed using eithersingle-stranded or double stranded DNA. Use of polymerase for primerextension requires a single-stranded DNA template. In preferredembodiments, the method of the present invention uses double-strandedDNA in order to obtain confirmatory opposite strand confirmation ofsequencing results. Double stranded DNA templates may be sequenced usingeither alkaline or heat denaturation to separate the two complementaryDNA templates into single strands. During polymerization, each moleculeof the DNA template is copied once as the complementary primer-extendedstrand. Use of thermostable DNA polymerases (e.g. Taq, Bst, Tth or VentDNA polymerase) enables repeated cycling of double-stranded DNAtemplates in the sequencing reaction through alternate periods of heatdenaturation, primer annealing, extension and dideoxy termination. Thiscycling process effectively amplifies small amounts of input DNAtemplate to generate sufficient template for sequencing.

Sequencing may also be performed directly on PCR amplification reactionproducts. Although the cloning of amplified DNA is relativelystraightforward, direct sequencing of PCR products facilitates andspeeds the acquisition of sequence information. As long as the PCRreaction produces a discrete amplified product, it will be amenable todirect sequencing. In contrast to methods where the PCR product iscloned and a single clone is sequenced, the approach in which thesequence of PCR products is analysed directly is generally unaffected bythe comparatively high error rate of Taq DNA polymerase. Errors arelikely to be stochastically distributed throughout the molecule. Thus,the overwhelming majority of the amplified product will consist of thecorrect sequence. Direct sequencing of PCR products has the advantageover sequencing cloned PCR products in that (1) it is readilystandardized because it is simple enzymatic process that does not dependon the use of living cells, and (2) only a single sequence needs to bedetermined for each sample.

As used herein, the terms “nucleic acid,” “polynucleotide,” and“oligonucleotide” refer to primers, probes, oligomer fragments to bedetected, oligomer controls and unlabeled blocking oligomers and shallbe generic to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), and to any other type ofpolynucleotide which is an N-glycoside of a purine or pyrimidine base,or modified purine or pyrimidine bases. There is no intended distinctionin length between the term “nucleic acid”, “polynucleotide” and“oligonucleotide”, and these terms are considered to be equivalent andinterchangeable, unless expressly indicated otherwise. These terms referonly to the primary structure of the molecule. Thus, these terms includedouble- and single-stranded DNA, as well as double- and single-strandedRNA. The oligonucleotide is comprised of a sequence of approximately atleast 6 nucleotides, preferably at least about 10-12 nucleotides, andmore preferably at least about 15-25 nucleotides corresponding to aregion of the designated nucleotide sequence. Nucleic acids may alsosubstitute standard nucleotide bases with nucleotide isoform analogs,including, but not limited to iso-C and iso-G bases, which may hybridizemore or less permissibly than standard bases, and which willpreferentially hybridize with complementary isoform analog bases. Manysuch isoform bases are described, for example, at www.idtdna.com. Theterm “corresponding to,” as used herein, as used herein to define anucleic acid sequence in terms of a reference nucleotide sequence, meansnucleotide sequences that match all or part of the reference sequence,and nucleotide sequences that are the complement of all or part of thereference sequence.

Oligonucleotides are not necessarily physically derived from anyexisting or natural sequence but may be generated in any manner,including chemical synthesis, DNA replication, reverse transcription ora combination thereof. The terms “oligonucleotide” or “nucleic acid”intend a polynucleotide of genomic DNA or RNA, cDNA, semisynthetic, orsynthetic origin which, by virtue of its origin or manipulation: (1) isnot associated with all or a portion of the polynucleotide with which itis associated in nature; and/or (2) is linked to a polynucleotide otherthan that to which it is linked in nature; and (3) is not found innature.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points toward the 5′ end of the other, theformer may be called the “upstream” oligonucleotide and the latter the“downstream” oligonucleotide.

The term “HR1,” as used herein refers to the heptad repeat 1 (HR1)region of the gp41 subunit of the HIV-1 env protein, represented bynucleotides 7388-7549 of SEQ ID NO:1.

The term “consisting essentially of,” as used herein in reference tospecified nucleotide sequences, means the specified sequences and anyadditional sequence that does not contain the predetermined lengthpolymorphisms. For example, the region consisting essentially of the HR1domain of HIV-1 env gp41 region, includes all or a part of HR1(nucleotides 7388-7549 of SEQ ID NO:1) and any additional nucleotidesequence of the adjacent 5′ and 3′ regions that do not include lengthpolymorphisms located outside the HR1 domain that may occur on asufficiently frequent basis among the population of prospective patientsto adversely affect the reliability of sequence results. Such lengthpolymorphisms may be located, for example, within approximately thefirst 25 or last 60 nucleotides of the gp41 domain. As used herein, aregion consisting essentially of the HR1 domain will preferablycorrespond to the region of nucleotides 7329-7614 of SEQ ID NO:1, orfragments thereof of 15 or more nucleotides. More preferably, the regionconsisting essentially of the HR1 domain will correspond to the regionof nucleotides 7388-7549 of SEQ ID NO:1 or fragments thereof of 15 ormore nucleotides. The region consisting essentially of the HR1 domainmay also correspond to the region of nucleotides 7492-7522 of SEQ IDNO:1 or fragments thereof of 15 or more nucleotides. It is understoodthat because the term “consisting essentially of” is used to definespecified sequences that exclude predetermined length polymorphisms, thepresent invention may include sequences that encompass other lengthpolymorphisms, whether known or unknown. The utility of the presentinvention arises primarily in situations where length polymorphisms giverise to a new quasispecies of a pathogenic vector, and that quasispeciesoccurs within the patient population with sufficient frequency that theclinical failure rate is unacceptably high. Thus, the present inventioncontemplates that the region containing the mutation of interest that issequenced, while “exclusive of the predetermined length polymorphisms,”may still include other length polymorphisms that, for example, areeither unknown or that do not occur within the patient population withsufficient frequency that the clinical failure rate has been determinedto be unacceptably high. It is sufficient, for purposes of the presentinvention, that even a single predetermined length polymorphisms hasbeen excluded from the region sequenced in order to enable complementarystrand confirmation of sequences from quasispecies of the vector definedby that predetermined length polymorphisms.

The term “encompass,” as used herein in reference to the location of aprimer relative to a reference location, means that a PCR extensionprimer is located so as to generate primer extension products thatinclude specified nucleotides or regions of nucleotides. The primer mayinclude nucleotide sequences that correspond to or are complementary toall or part of the reference location. Alternatively, the primer may becomplementary to a region located 3′ of the reference location.

The term “primer” may refer to more than one primer and refers to anoligonucleotide, whether occurring naturally, as in a purifiedrestriction digest, or produced synthetically, which is capable ofacting as a point of initiation of synthesis along a complementarystrand when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand iscatalyzed. Such conditions include the presence of four differentdeoxyribonucleoside triphosphates and a polymerization-inducing agentsuch as DNA polymerase or reverse transcriptase, in a suitable buffer(“buffer” includes substituents which are cofactors, or which affect pH,ionic strength, etc.), and at a suitable temperature.

The primer is preferably single stranded for maximum efficiency inamplification, but may alternatively be double stranded. If doublestranded, the primer is first treated to separate its strands beforebeing used to prepare extension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the agent forpolymerization. The exact lengths of the primers will depend on manyfactors, including temperature and source of primer and use of themethod. For example, depending on the complexity of the target sequence,the oligonucleotide primer typically contains 10-50, and preferably15-25 nucleotides, although it may contain more or fewer nucleotides.Short primer molecules generally require lower temperatures to formsufficiently stable hybrid complexes with the template.

The term “extension primer,” as used herein, means a polynucleotidesequence that is complementary to a template sequence, and which iscapable of hybridizing to and extending a sequence under polymerasechain reaction conditions to produce a primer extension product.

The term “complement,” and its related adjective form “complementary,”when used in reference to two nucleic acid sequences, means that whentwo nucleic acid sequences are aligned in anti-parallel association(with the 5′ end of one sequence paired with the 3′ end of the othersequence) the corresponding G and C nucleotide bases of the sequencesare paired, and the corresponding A and T nucleotide bases are paired.Certain bases not commonly found in natural nucleic acids may beincluded in the nucleic acids of the present invention and include, forexample, inosine and 7-deazaguanine.

The term “allele,” used herein, means a specific version of a nucleotidesequence at a polymorphic genetic locus.

The term “polymorphic site,” as used herein means a given nucleotidelocation in a genetic locus which is variable within a population.

The term “genetic locus,” as used herein means a specific position orlocation of a nucleotide or region of nucleotides in a DNA sequence orin the corresponding RNA strand packaged in a viral particle, which isderived from the transcribed DNA sequence.

The nucleotides adenosine, cytosine, guanine and thymine are representedby their one-letter codes A, C, G, and T respectively. Inrepresentations of degenerate primers, the symbol R refers to either Gor A, the symbol Y refers to either T/U or C, the symbol M refers toeither A or C, the symbol K refers to either G or T/U, the symbol Srefers to G or C, the symbol W refers to either A or T/U, the symbol Brefers to “not A”, the symbol D refers to “not C”, the symbol H refersto “not G”, the symbol V refers to “not T/U” and the symbol N refers toany nucleotide. In the specification and claims of this application, adegenerate primer refers to any or all of the combinations of basechoices and to either DNA or the corresponding RNA sequence (i.e., withT replaced by U). Thus, a degenerate primer may represent a singlespecies, or a mixture of two species which fall within the choices, or amixture of three choices which fall with the choices, and so on up to amixture containing all the possible combinations. Isoform nucleotidebases are represented using nomenclature generally accepted by those inthe art.

The term “oligonucleotide primer,” as used herein, means a moleculecomprised of more than three deoxyribonucleotides or ribonucleotides.Its exact length will depend on many factors relating to the ultimatefunction and use of the oligonucleotide primer, including temperature ofthe annealing reaction, and the source and composition of the primer.Amplification primers must be sufficiently long to prime the synthesisof extension products in the presence of the agent for polymerization.The oligonucleotide primer is capable of acting as an initiation pointfor synthesis when placed under conditions which induce synthesis of aprimer extension product complementary to a nucleic acid strand. Theconditions can include the presence of nucleotides and an inducing agentsuch as a DNA polymerase at a suitable temperature and pH. In preferredembodiments, the primer is a single-stranded oligodeoxyribonucleotide ofsufficient length to prime the synthesis of an extension product from aspecific sequence in the presence of an inducing agent. In one aspect ofthe present invention, the oligonucleotide primers are from about 10 toabout 50 nucleotides long, and preferably from about 15 to about 30nucleotides long, although a primer may contain more or fewernucleotides. The oligonucleotide primers are generally at least 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides long. More preferably,primers will contain around 20-25 nucleotides. Sensitivity andspecificity of the oligonucleotide primers are determined by the primerlength and uniqueness of sequence within a given sample of templatenucleic acid. Primers which are too short, for example, may shownon-specific binding to a wide variety of sequences.

The oligonucleotide primers used in the present invention may alsoinclude universal primers. Universal primers are used for convenience inamplifying and sequencing a polynucleotide sequence that has beeninserted into a standard vector that contains sequences complementary tothe universal primers. Universal sequencing primers are well-known tothose in the art, and include, for example, primers referred to as T7,SP6, M13(−40), M13(−20), and M13/pUC. An amplification primer may bedesigned so as to include the complement of a universal primer, so thatthe amplification product (the amplicon) incorporates a universal primersite, thereby facilitating subsequent sequencing using complementaryuniversal sequencing primers.

The primers of the present invention may also include random additionalsequence between the primer sequence and the sequence of interest tofacilitate more accurate sequencing. Because the initial 10-50 basepairs of a sequence are typically unreadable, the addition of a 10-50base pair random sequence shifts the critical sequence downstream of theinitial unreadable sequence so that the sequence of interest is locatedwithin the region where accurate reading of the sequence occurs.

The term “reverse transcription” means the process of generating a DNAcomplement to an RNA molecule, and is generally accomplished with theuse of a reverse transcriptase enzyme. A primer may be used to initiatepolymerization; this primer may be one of a primer pair later used forPCR amplification. The RNA molecule is then separated from the copiedDNA (“cDNA”) or degraded by an RNAse H activity of an enzyme thusallowing the second strand of cDNA to be generated by a templatedependent DNA polymerase. This method is disclosed in Units 3.7 and 15.4of Current Protocols in Molecular Biology, Eds. Ausubel, F. M. et al,(John Wiley & Sons; 1995), the contents of which are incorporated hereinby reference.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA techniques, oligonucleotide synthesis which are withinthe skill of the art. Such techniques are explained fully in theliterature. Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); NucleicAcid Hybridization (B. D. Hames & S. J. Higgins, eds., 1984); APractical Guide to Molecular Cloning (B. Perbal, 1984); and a series,Methods in Enzymology (Academic Press, Inc.), the contents of all ofwhich are incorporated herein by reference.

Methods of the Invention

The present invention is directed to a method for detecting the presenceor absence of a mutation of interest that is adjacent to a differentpredetermined length polymorphism. The methods and reagents of thepresent invention are most advantageously used for obtainingpolynucleotide sequence of an exogenous vector, such as an infectiouspathogen, in a patient sample that may contain multiple quasispecies ofthe vector relative to the length polymorphism. The presence of mixedlength polymorphisms among quasispecies in a single patient sampleprecludes confirmation of the sequence results from the complementaryopposite strand DNA of the vector where the region that is sequencedencompasses the length polymorphism. Generally, the method of thepresent invention comprises selectively sequencing both complementarystrands of the region associated with the mutation of interest,exclusive of the adjacent predetermined length polymorphism, andcomparing the sequence of the complementary strands to confirm that thesequence of each strand is the complement of the other strand at allnucleotide bases.

While the methods of the present invention were developed initially withrespect to the HIV-1 gene, such methods are nevertheless applicable toany pathogen having length polymorphisms among multiple quasispecies.Accordingly, the present invention is directed to a method for detectingthe presence or absence of a mutation of interest in the nucleic acid ofa pathogen, such as HIV-1, wherein the mutation of interest is locatedadjacent to a length polymorphism defining multiple quasispecies of thepathogen, comprising:

-   -   a) obtaining from the patient sample a double-stranded DNA        template encompassing the mutation of interest;    -   b) sequencing a first strand of a region of the DNA template        containing the mutation of interest, exclusive of the length        polymorphism;    -   c) sequencing a second strand of a region of the DNA template        containing the mutation of interest, exclusive of the length        polymorphism; and    -   d) comparing the sequence of the first strand with the sequence        of the second strand to obtain complementary strand confirmation        of the sequence of the mutation of interest.        Sequencing in steps (b) and (c) may be performed using any        suitable DNA template capable of yielding genetic sequence        information of the specified region of the exogenous vector.

In another aspect, the present invention is directed to a method fordetecting the presence or absence of a mutation of interest in thenucleic acid of a pathogen, such as HIV-1, wherein the mutation ofinterest is located adjacent to a length polymorphism defining multiplequasispecies of the pathogen, comprising:

-   -   a) obtaining from the patient sample a double-stranded DNA        template encompassing the mutation of interest;    -   b) amplifying a first region of the DNA template containing both        the mutation of interest and the length polymorphism;    -   c) sequencing a first strand of a region of the DNA template        containing the mutation of interest, exclusive of the length        polymorphism;    -   d) sequencing a second strand of a region of the DNA template        containing the mutation of interest, exclusive of the length        polymorphism; and    -   e) comparing the sequence of the first strand with the sequence        of the second strand to obtain complementary strand confirmation        of the sequence of the mutation of interest.        In this method, the amplification step includes the mutation of        interest and the length polymorphism, and it is in the        sequencing steps (c) and (d) that the length polymorphism is        excluded.

In yet another aspect, the present invention is directed to a method fordetecting the presence or absence of a mutation of interest in thenucleic acid of a pathogen, such as HIV-1, wherein the mutation ofinterest is located adjacent to a length polymorphism defining multiplequasispecies of the pathogen, comprising:

-   -   a) obtaining from the patient sample a double-stranded DNA        template encompassing the mutation of interest;    -   b) amplifying a first genetic region of the DNA template        comprising the mutation of interest, exclusive of the length        polymorphism;    -   c) sequencing a first strand of a region of the DNA template        containing the mutation of interest, exclusive of the length        polymorphism;    -   d) sequencing a second strand of a region of the DNA template        containing the mutation of interest, exclusive of the length        polymorphism; and    -   e) comparing the sequence of the first strand with the sequence        of the second strand to obtain complementary strand confirmation        of the sequence of the mutation of interest.        In this embodiment, it is in the amplification step that the        length polymorphism is excluded, and subsequent sequencing of        the amplified region or a portion of the amplified region also        excludes the length polymorphism.

In a one aspect of the invention, the mutation of interest is located ina region of HIV selected from the group consisting of: gp41 and gp120.In another aspect of the invention, the mutation of interest is locatedin the HIV-1 envelope glycoprotein gp41. In yet another aspect of theinvention, the mutation of interest is located within positions 36-45gp41.

As illustrated by the above embodiments, the common feature of thevarious embodiments of the present invention is the exclusion of thelength polymorphism within the region of the DNA that is sequenced,permitting subsequent comparison of the first strand sequence with thesecond strand sequence to obtain complementary strand confirmation ofthe sequence of the mutation of interest, without confounding results asa consequence of having multiple length polymorphism quasispeciespresent having different nucleic acid lengths. It will be understood bythose in the art that various approaches may be utilized prior to thesequencing step to provide a DNA template that can yield sequence dataexclusive of the length polymorphism.

Source of DNA

In one aspect of the invention, the method comprises first obtainingfrom the patient sample a double-stranded polynucleotide templateencompassing the mutation of interest. The double-strandedpolynucleotide template may initially comprise genomic DNA or a fragmentof genomic DNA. This template will encompass not only the mutation ofinterest, but may also encompass the region containing the lengthpolymorphism giving rise to multiple quasispecies.

A double-stranded polynucleotide template will typically be preparedfrom a patient sample by treating a patient sample containing DNA so asto make all or a portion of the DNA in the sample accessible forhybridization with oligonucleotide primers, for example by lysis,centrifugation to remove cellular debris and proteolytic digestion toexpose the DNA. The DNA template may therefore contain only nuclear DNA,only mitochondrial DNA, or some sub-fraction of nuclear or mitochondrialDNA obtained by isolation from a tissue sample. The DNA template mayalso be prepared by conversion, for example by reverse transcription, ofa total mRNA preparation or the genome of an RNA virus to cDNA; DNAisolated from an individual bacterial colony growing on a plate or froman enriched bacterial culture; and a viral DNA preparation wheresubstantially the entire viral genome is isolated.

DNA can be prepared from fluid samples, e.g., blood or urine or tissuesamples by any of a number of techniques, including lysis,centrifugation to remove cellular debris and proteolytic digestion toexpose the DNA; salt precipitation or standard SDS-proteinase K-phenolextraction. Samples can also be prepared using kits, for example thePure Gene DNA Isolation Kit (Gentra).

Amplification of Nucleic Acids

Preferred embodiments of the present invention include the step ofamplifying DNA to provide an abundant source of DNA for subsequentsequencing. In one aspect, the method of the present inventionoptionally comprises amplifying a first region of the DNA templatecontaining both the mutation of interest and the length polymorphism. Anamplification product that contains the predetermined lengthpolymorphism will subsequently be sequenced using primers that excludethe length polymorphism.

Alternatively, in another aspect, the method may comprise selectivelyamplifying a first genetic region of the DNA template containing theregion containing the mutation of interest, exclusive of the lengthpolymorphism. An amplification product that excludes the predeterminedlength polymorphism may be sequenced directly, using the same primerscorresponding to the amplification primers, using primers complementaryto a different region of the amplified fragment, or using universalprimers complementary to a universal primer template incorporated intothe amplification product during PCR (using primers that include theuniversal primer).

Typically, prior to sequencing, a sequencing template is prepared byfirst amplifying a region of DNA that encompasses the target region tobe sequenced. It is not necessary that the sequence to be amplified bepresent initially in a pure form; it may be a minor fraction of acomplex mixture or a portion of nucleic acid sequence. The startingnucleic acid may contain more than one desired specific nucleic acidsequence which may be the same or different. Therefore, the presentprocess is useful not only for producing large amounts of one specificnucleic acid sequence, but also for amplifying simultaneously more thanone different specific nucleic acid sequence located on the same ordifferent nucleic acid molecules if more than one of the base pairvariations in sequence is present.

In one aspect, the present invention is directed to amplification andsequencing primers used in a method for genotyping HIV-1 env. The methodutilizes well-known methods for amplifying specific nucleic acidsequences using the technique of polymerase chain reaction (or PCR) orsome other primer extension based methodology. Polymerase chain reaction(PCR) is very widely known in the art. For example, U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159; K. Mullis, Cold Spring Harbor Symp.Quant. Biol., 51:263-273 (1986); and C. R. Newton & A. Graham,Introduction to Biotechniques: PCR, 2.sup.nd Ed., Springer-Verlag (NewYork: 1997), the disclosures of which are incorporated herein byreference, describe processes to amplify a nucleic acid sample targetusing PCR amplification extension primers which hybridize with thesample target. As the PCR amplification primers are extended, using aDNA polymerase (preferably thermostable), more sample target is made sothat more primers can be used to repeat the process, thus amplifying thesample target sequence. Typically, the reaction conditions are cycledbetween those conducive to hybridization and nucleic acidpolymerization, and those that result in the denaturation of duplexmolecules.

To briefly summarize, in the first step of the reaction, the nucleicacid molecules of the sample are transiently heated, and then cooled, inorder to denature double stranded molecules. Forward and reverse primersare present in the amplification reaction mixture at an excessconcentration relative to the sample target. When the sample isincubated under conditions conducive to hybridization andpolymerization, the primers hybridize to the complementary strand of thenucleic acid molecule at a position 3′ to the sequence of the regiondesired to be amplified that is the complement of the sequence whoseamplification is desired. Upon hybridization, the 3′ ends of the primersare extended by the polymerase. The extension of the primer results inthe synthesis of a DNA molecule having the exact sequence of thecomplement of the desired nucleic acid sample target. The PCR reactionis capable of exponentially amplifying the desired nucleic acidsequences, with a near doubling of the number of molecules having thedesired sequence in each cycle. Thus, by permitting cycles ofhybridization, polymerization, and denaturation, an exponential increasein the concentration of the desired nucleic acid molecule can beachieved.

Preparation of Nucleic Acid Amplification Templates

The present invention is directed to methods of amplifying andsequencing pathogens, including HIV-1 env and its variant forms. Themethod of the present invention may employ, for example, DNA or RNA,including messenger RNA, which DNA or RNA may be single stranded ordouble stranded. In addition, a DNA-RNA hybrid which contains one strandof each may be utilized. A mixture of any of these nucleic acids mayalso be employed, or the nucleic acids produced from a previousamplification reaction herein using the same or different primers may beso utilized. The specific nucleic acid sequence to be amplified may beonly a fraction of a larger molecule or can be present initially as adiscrete molecule, so that the specific sequence constitutes the entirenucleic acid.

It is not necessary that the sequence to be amplified be presentinitially in a pure form; it may be a minor fraction of a complexmixture or a portion of nucleic acid sequence. The starting nucleic acidmay contain more than one desired specific nucleic acid sequence whichmay be the same or different. Therefore, the present process is usefulnot only for producing large amounts of one specific nucleic acidsequence, but also for amplifying simultaneously more than one differentspecific nucleic acid sequence located on the same or different nucleicacid molecules if more than one of the base pair variations in sequenceis present.

The nucleic acid templates may be obtained from any source, for example,from plasmids such as pBR322, from cloned DNA or RNA, or from naturalDNA or RNA from any source, such as HIV infected plasma or serumobtained from patients. DNA or RNA may be extracted from blood, tissuematerial or amniotic cells by a variety of techniques such as thatdescribed by Maniatis et al., Molecular Cloning (1982), 280-281.

The cells may be directly used without purification of the nucleic acidif they are suspended in hypotonic buffer and heated to about 90°-100°C., until cell lysis and dispersion of intracellular components occur,generally about 1 to 15 minutes. After the heating step theamplification reagents may be added directly to the lysed cells. Thisdirect cell detection method may be used on peripheral blood lymphocytesand amniocytes.

The target nucleic acid contained in the sample will initially be in theform of RNA, and is preferably reverse transcribed into cDNA, and thendenatured, using any suitable denaturing method, including physical,chemical, or enzymatic means, which are known to those of skill in theart. A preferred physical means for strand separation involves heatingthe nucleic acid until it is completely (>99%) denatured. Typical heatdenaturation involves temperatures ranging from about 80° C. to about105° C., for times ranging from a few seconds to minutes. As analternative to denaturation, the target nucleic acid may exist in asingle-stranded form in the sample, such as, for example,single-stranded RNA or DNA viruses.

The denatured nucleic acid strands are then incubated with preselectedoligonucleotide primers, and, optionally, a labeled oligonucleotide(referred to herein as a “probe”) for purposes of detecting theamplified sequence) under conditions that facilitate the binding of theprimers and probes to the single nucleic acid strands. As known in theart, the primers are selected so that their relative positions along aduplex sequence are such that an extension product synthesized from oneprimer, when the extension product is separated from its template(complement), serves as a template for the extension of the other primerto yield a replicate chain of defined length.

Sequencing of Nucleic Acids

Amplification of DNA as described above will result in an abundantsource of DNA for sequencing. The polynucleotide templates prepared asdescribed above are sequenced using any of the numerous methodsavailable and known to those in the art for sequencing nucleotides.

In preferred aspects of the present invention, the DNA template used forsequencing will be double stranded. Double stranded DNA permitssimultaneous sequencing of complementary strands, enabling confirmationof correct sequence results by comparison of the sequence obtained fromeach strand, which should be exactly complementary.

The amplification methods used in the present invention may also besimultaneously used in conjunction with sequencing. Methods forsimultaneous amplification and sequencing are widely known in the art,and include coupled amplification and sequence (CAS) (described by Ruanoand Kidd, Proc. Nat'l. Acad. Sci. (USA) 88(7): 2815-2819 (1991), and inU.S. Pat. No. 5,427,911, which are incorporated herein by reference),and CLIP amplification and sequencing (described in U.S. Pat. No.6,007,983, and in J. Clin. Microbiology 41(4); 1586-1593 (April 2003)which are incorporated herein by reference). CLIP sequencing subjectsPCR amplification fragments previously generated to simultaneous PCRamplification and direct sequencing. In CAS sequencing, a sample istreated in a first reaction stage with two primers and amplified for anumber of cycles to achieve 10,000 to 100,000-fold amplification. AddNTP is then added during the exponential phase of the amplificationreaction, and the reaction is processed for additional thermal cycles toproduce chain-terminated sequencing fragments. The CAS process requiresan intermediate addition of reagents (the ddNTP reagents), whichintroduces opportunity for error or contamination and increases thecomplexity of any apparatus which would be used for automation. The CASmethodology is therefore preferably combined with CLIP sequencing, whichsubjects PCR amplification fragments previously generated tosimultaneous PCR amplification and direct sequencing. Simultaneousamplification and sequencing using the CLIP® method may be accomplished,for example, using the reagents and conditions described and provided incommercially available kits, such as the TRUGENE® HIV genotyping kit(Bayer HealthCare LLC).

In particular aspects, the present invention relates to sequencing ofinfectious pathogens, such as bacteria or viruses, such as Hepatitis B,Hepatitis C, and Human Immunodeficiency Virus, in particular HIV-1 env,and their variant forms. The double stranded DNA template used in themethod of the present invention may be derived from, for example, DNA orRNA, including messenger RNA, which may be single stranded or doublestranded. In addition, the DNA template may be in the form of a DNA-RNAhybrid which contains one strand of DNA and one strand of RNA. A mixtureof any of these nucleic acids may also be employed, or the nucleic acidsproduced from a previous amplification reaction herein using the same ordifferent primers may be so utilized. The specific nucleic acid sequenceto be amplified may be only a fraction of a larger molecule or can bepresent initially as a discrete molecule, so that the specific sequenceconstitutes the entire nucleic acid.

Sequencing the HR1 and/or HR2 Domains of HIV-1 gp41

The present invention includes a novel method and reagents forgenotyping the HIV-1 transmembrane glycoprotein (gp41) in a samplesuspected of containing the HIV-1 virus.

The present invention addresses the above-mentioned problem, byproviding primers that encompass all or part of the HR1 and HR2 domainsof gp41, or all or part of the HR1 domain of gp41.

The sequencing primers of the present invention consist ofoligonucleotides specific to HIV-1, which can be used to amplify andsequence a portion of gp41 DNA. In accordance with methods known tothose in the art, a sample obtained from an individual suspected ofbeing infected with the HIV-1 virus is used to recover viral RNA, eitherin the form of RNA or DNA. Viral HIV-1 RNA obtained from the sample isreverse transcribed to cDNA. The cDNA template is then amplified, usingPolymerase Chain Reaction or some other primer extension based method.The resulting amplified fragment is then initially sequenced with a setof primers encompassing the gp41 subunit, encompassing the HR1 and HR2domains, or alternatively encompassing the HR1 domain, using cyclesequencing methods or CLIP™ bi-directional sequencing.

One particular aspect of the present invention is a method foramplifying and genotyping the gp41 subunit of the HIV-1 env gene in asample suspected of containing the HIV-1 virus, comprising (1)amplifying the HIV-1 env region, (2) determining sequence of a regionencompassing both the HR1 and HR2 domains of gp41.

The present invention is generally directed to a novel method andreagents for sequencing and genotyping the HR1 domain of the HIV-1transmembrane glycoprotein (gp41) in a sample suspected of containingthe HIV-1 virus. The higher complexity of the gp41 region surroundingthe first heptad repeat domain (HR1) was found to be due to relativelyfrequent occurrence of insertion or deletion mutations near the HR1domain. Mutations in the HR1 domain are known to occur, for example, atpositions 36 to 38 of the HIV-1 envelope glycoprotein gp41. Particularmutations include G36V/D/S, I37V, V38A/M/E, Q39R, Q40H, N42T,N43H/E/D/S, L44M, L45M. Mixtures of HIV-1 viral populations with lengthpolymorphisms were found to occur in plasma, resulting in inconsistentHR1 nucleotide sequence data and conflicting bi-directional sequencedata. This problem was not previously recognized, possibly because themethods have relied upon cloning of RT-PCR products before sequencing orbecause uni-directional data has been accepted without confirmatorybi-directional data. The present invention addresses the above-mentionedproblem, by providing a method and primers for sequencing a regionconsisting essentially of the HR1 domain of HIV-1 env, but excluding theregions of higher variability responsible for length polymorphisms. Theprimers of the present invention are therefore useful for confirmingsequence for samples that cannot be accurately characterized usingsequencing primers that attempt to sequence through the regioncontaining length polymorphisms before reaching the HR1 domain.

Sequencing Primers for gp41

The sequencing primers of the present invention include both forwardprimers and reverse primers, which may be labeled with a detectablelabel. For most common sequencing instruments, a fluorescent label isdesirable, although other labels types including colored, chromogenic,fluorogenic (including chemiluminescent) and radiolabels could also beemployed. The primer combination may include other reagents appropriatefor reverse transcription, amplification or sequencing, and may, ofcourse, include HIV-1 genetic material for analysis.

In one aspect, the present invention includes a method and primers fordetermining the sequence of the HR1 and HR2 domains of gp41. In aparticular embodiment, present invention includes a method and primersfor determining the sequence of both DNA strands (bi-directional) of theregion encompassing the HR1 and HR2 domains of gp41.

In another aspect, the present invention also includes methods andprimers for sequencing a region consisting essentially of the HR1 domainof the HIV-1 glycoprotein gp41. The sequencing primers of the presentinvention may be any suitable sequencing primer having the desiredspecificity to sequence a region consisting essentially of the HR1domain of HIV-1. In accordance with methods known to those in the art, asample obtained from an individual known to be or suspected of beinginfected with the HIV-1 virus is used to recover viral RNA, either inthe form of RNA or DNA. Viral HIV-1 RNA obtained from the sample isreverse transcribed to cDNA. The cDNA template is then amplified, usingPolymerase Chain Reaction or some other primer extension based method.In preferred embodiments, the amplified region encompasses the regionencoding the HIV-1 env protein, although broader or narrower regions arealso suitable for use as a template for sequencing specific regions ofthe HIV env domain. The resulting amplified fragment is then sequenced(for example, by cycle sequencing or CLIP® bi-directional sequencing)with a set of primers or, in the case of uni-directional sequencing, asingle primer, that provides sequence for a region consistingessentially of the HR1 domain, but which does not sequence throughregions of length polymorphisms caused by insertion and/or deletionmutations near the HR1 domain prior to sequencing the HR1 region itself.The primers of the present invention are thus complementary to regionssituated between the regions of length polymorphisms caused by insertionand/or deletion mutations and the HR1 domain itself, so that extensionof the primer proceeds in the direction away from the lengthpolymorphisms towards the HR1 domain. In a particular embodiment, thepresent invention provides a method for sequencing the HR1 domain of thegp41 subunit of the HIV-1 env gene in a sample suspected of containingthe HIV-1 virus, comprising determining the nucleotide sequence of aregion consisting essentially of the HR1 domain of gp41, but excludingregions adjacent to the HR1 domain that include mutations associatedwith length polymorphisms.

The primers of the present invention enable determination of thesequence of HR1 in clinical settings where other primers result insequence data that is difficult to interpret as a result of lengthpolymorphisms among mixed plasma subpopulations of HIV-1. These primersmay be utilized to provide confirmation of HR1 mutations, but may alsobe used as an alternative sequencing primer set for HR1 only coverage.

The primers of the present invention may be a single primer for use indetermining the sequence of a single strand of DNA. Alternatively, in apreferred embodiment of the invention, the primers are a combination ofprimers for determining the sequence of both DNA strands (bi-directionalsequence) of the region encompassing the HR1 domain of gp41. Thesequencing primers of the present invention may therefore includeforward primers, reverse primers, or both forward and reverse primers.When used for clinical purposes in determining the genotype of HIV-1from a patient sample, it is desirable to obtain sequence of bothforward and reverse strands of DNA, thereby obtaining confirmation ofsequence results. It is understood, however, that the sequencing primersof the present invention may be used when only obtaining sequence of oneor the other of the forward and reverse strands.

The primers of the present invention are specific to regions betweendrug resistance associated mutations within HR1 and length polymorphismsfound among mixed plasma subpopulations of HIV. In functional terms,such primers can be defined as primers that are capable of generatingsequencing reaction products that do not include insertion or deletionmutations associated with length polymorphisms found among mixed plasmasubpopulations of HIV. Because the objective is to obtain consistentsequence data for the HR1 region (specifically, the drug resistancemutations located within HR1), it is contemplated that primers willinitiate extension in the direction that first provides sequence datafor the HR1 region (away from length polymorphisms located in theopposite direction of extension), followed by sequence that may includelength polymorphisms on the other side of HR1. By first generatingsequence through the HR1 region (prior to generating sequence throughthe region of length polymorphisms), consistent nucleotide sequence datafor the HR1 region is generated. The existence of confounded sequenceresulting from the length polymorphisms following the HR1 region doesnot therefore adversely affect the sequence results of the HR1 regionitself. It is therefore understood that the primers may sequence througha region of length polymorphism, provide such sequence is generatedafter the sequence for HR1 has been generated first.

Examples of acceptable sequencing primers are disclosed below. Althoughthe sequencing primers of the present invention are preferably selectedfrom among primers having the same sequence as disclosed below, it iscontemplated that the present invention includes degenerate sequenceshaving specificity for major HIV-1 subtypes, but which may also havespecificity for less common sub-types. The design and construction ofsuch degenerate sequences is well know to those in the art.

Generally, DNA sequencing primers consist of 15 or more nucleotidebases, preferably from 18 to 30 nucleotide bases. The sequencing primersmay also include fragments of the above primers having 18, 19, 20, 21,22, 23, 24, or 25 nucleotides. Preferably, primers have a meltingtemperature (Tm) in the range of 52° C. to 65° C., particularly iftemplates are GC rich, since this can lead to secondary primingartifacts and noisy sequences. In addition, preferred primers will notdimerize or form significant hairpins, and will lack secondary primingsites. Also, primers will have low specific binding at the 3′ end (i.e.,will have a lower GC content, preferably from 40-60%, to avoidmispriming. Computer software is available to design primers with thesecharacteristics, which includes LaserGene (DNAStar), Oligo (NationalBiosciences, Inc.), MacVector (Kodak/IBI) and the GCG suite. Inaddition, primers may be designed to satisfy the above criteria usingthe Whitehead Institute PCR primer program, available athttp://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi. Additionaldesign criteria is known and available to those skilled in the art.

Because sequencing primers, as opposed to amplification primers, may notbe mixed together if they do not have the same location for the 3′ base,specific degenerate base positions are illustrated below, although it isto be understood that the 3′ and 5′ locations may be modified. The 5′nucleotide location may be changed to include regions of greatersequence conservation or to modify melting temperature and stringency ofbinding. A non-degenerate primer set is preferred, provided the successrate is sufficient to obtain sequence for the desired HIV-1 subtypes.Reaction conditions may also be adjusted to optimize performance.Examples of potential modifications of sequencing primers are disclosedbelow.

In a particular embodiment, the region encompassing both the HR1 and HR2domains of gp41 is sequenced using sequencing primers selected from thefollowing:

SEQ ID NO:2 5′-GCACCXACSARGGCAAAGAGAMGAGYGG-3′ SEQ ID NO:35′-ACCAAGGCAAAGAGAAGAGTGG-3′ SEQ ID NO:4 5′-CCCACCAAGGCAAAGAGAAGAGTGG-3′SEQ ID NO:5 5′-GCACCCACCAAGGCAAAGAGAAGAGTGG-3′ SEQ ID NO:65′-GCACCCACCAAGGCAAAGAGAAGAGYGG-3′ SEQ ID NO:75′-GCACCCACCAAGGCAAAGAGAMGAGYGG-3′ SEQ ID NO:85′-GCACCCACCAGGGCAAAGAGAMGAGYGG-3′ SEQ ID NO:95′-GCACCCACGAGGGCAAAGAGAMGAGYGG-3′ SEQ ID NO:105′-GCACCNACGAGGGCAAAGAGAMGAGYGG-3′ SEQ ID NO:115′-ACGAGGGCAAAGAGAMGAGYGG-3′ SEQ ID NO:12 5′-CCAAGGCAAAGAGAAGAGTG-3′ SEQID NO:13 5′-CGAGGGCAAAGAGAMGAGYG-3′ SEQ ID NO:145′-GCACCCACCAAGGCAAAGAGAAGAGTGG-3′ SEQ ID NO:155′-ACCAAGGCAAAGAGAAGAGTGG-3′ SEQ ID NO:165′-tartaggaggnttrataggnttaagaata-3′ SEQ ID NO:173′-gtaggaggcttgataggtttaag-5′ SEQ ID NO:183′-tagtaggaggcttgataggtttaag-5′ SEQ ID NO:193′-tartaggaggcttgataggtttaag-5′ SEQ ID NO:203′-tartaggaggnttgataggtttaag-5′ SEQ ID NO:213′-tartaggaggnttrataggtttaag-5′ SEQ ID NO:223′-tartaggaggnttrataggnttaag-5′ SEQ ID NO:233′-tartaggaggnttrataggnttaagaata-5′ SEQ ID NO:243′-ggaggnttrataggnttaagaata-5′ SEQ ID NO:253′-ggvggnttrataggnttaagaata-5′ SEQ ID NO:26 3′-taggaggnttrataggnttaag-5′SEQ ID NO:27 3′-ggaggcttggtaggtttaaga-5′ SEQ ID NO:283′-ggvggnttrataggnttaaga-5′ SEQ ID NO:293′-tagtaggaggcttgataggtttaagaata-5′ SEQ ID NO:303′-gtaggaggcttgataggtttaag-5′

The sequencing primers may include, for example, at least one forwardsequencing primer selected from the group consisting of the following:

SEQ ID NO:2 5′-GCACCXACSARGGCAAAGAGAMGAGYGG-3′ SEQ ID NO:35′-ACCAAGGCAAAGAGAAGAGTGG-3′ SEQ ID NO:4 5′-CCCACCAAGGCAAAGAGAAGAGTGG-3′SEQ ID NO:5 5′-GCACCCACCAAGGCAAAGAGAAGAGTGG-3′ SEQ ID NO:65′-GCACCCACCAAGGCAAAGAGAAGAGYGG-3′ SEQ ID NO:75′-GCACCCACCAAGGCAAAGAGAMGAGYGG-3′ SEQ ID NO:85′-GCACCCACCAGGGCAAAGAGAMGAGYGG-3′ SEQ ID NO:95′-GCACCCACGAGGGCAAAGAGAMGAGYGG-3′ SEQ ID NO:105′-GCACCNACGAGGGCAAAGAGAMGAGYGG-3′ SEQ ID NO:115′-ACGAGGGCAAAGAGAMGAGYGG-3′ SEQ ID NO:12 5′-CCAAGGCAAAGAGAAGAGTG-3′ SEQID NO:13 5′-CGAGGGCAAAGAGAMGAGYG-3′ SEQ ID NO:145′-GCACCCACCAAGGCAAAGAGAAGAGTGG-3′

In one embodiment of the present invention, the set of bi-directionalprimary sequencing primers includes a first primer comprising thenucleotide sequence of SEQ ID NO:2, and fragments thereof of 15 or morenucleotides, inclusive of the 3′ terminus. SEQ ID NO:2 describes a setof degenerate primers that may be used as the forward primer, where X is5-nitroindole or C, S represents G or C, R represents G or A, Mrepresents A or C, and Y represents T or C. Primers degenerate at Y(T/C)are designed to detect rare isolates of both the B and A HIV subtypes.Similarly, primers degenerate at M(A/C) are designed to detect F-typeHIV recombinants. Other variations of the above sequences may beutilized to permit detection of other HIV variants.

In another embodiment, the present invention includes a set ofbi-directional primary sequencing primers that includes a first primerselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, and fragments thereof of15 or more nucleotides.

In yet another embodiment, the present invention includes a set ofbi-directional primary sequencing primers that includes a first primercomprising the sequence of SEQ ID NO:14, and fragments thereof of 15 ormore nucleotides.

In yet another embodiment, the present invention includes a set ofbi-directional primary sequencing primers includes a first primercomprising the sequence of SEQ ID NO:3.

The primary sequencing primers may also include at least one reversesequencing primer selected from the group consisting of the following:

SEQ ID NO:15 5′-ACCAAGGCAAAGAGAAGAGTGG-3′ SEQ ID NO:165′-tartaggaggnttrataggnttaagaata-3′ SEQ ID NO:173′-gtaggaggcttgataggtttaag-5′ SEQ ID NO:183′-tagtaggaggcttgataggtttaag-5′ SEQ ID NO:193′-tartaggaggcttgataggtttaag-5′ SEQ ID NO:203′-tartaggaggnttgataggtttaag-5′ SEQ ID NO:213′-tartaggaggnttrataggtttaag-5′ SEQ ID NO:223′-tartaggaggnttrataggnttaag-5′ SEQ ID NO:233′-tartaggaggnttrataggnttaagaata-5′ SEQ ID NO:243′-ggaggnttrataggnttaagaata-5′ SEQ ID NO:253′-ggvggnttrataggnttaagaata-5′ SEQ ID NO:26 3′-taggaggnttrataggnttaag-5′SEQ ID NO:27 3′-ggaggcttggtaggtttaaga-5′ SEQ ID NO:283′-ggvggnttrataggnttaaga-5′] SEQ ID NO:293′-tagtaggaggcttgataggtttaagaata-5′] SEQ ID NO:303′-gtaggaggcttgataggtttaag-5′

In one embodiment of the present invention, the set of bi-directionalprimary sequencing primers includes a second primer comprising thenucleotide sequence of SEQ ID NO:16, and fragments thereof of 15 or morenucleotides, inclusive of the 3′ terminus. SEQ ID NO:16 describes a setof degenerate primers that may be used as the forward primer, where Rrepresents G or A, X represents 5-nitroindole or C, Z represents5-nitroindole or T, and V represents A or G or C, but not T. Othervariations of the above sequences may be utilized to permit detection ofother HIV variants.

In another embodiment, the present invention includes a set ofbi-directional primary sequencing primers that includes a second primerselected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

In yet another embodiment, the present invention includes a set ofbi-directional primary sequencing primers that includes a second primercomprising the sequence of SEQ ID NO:29, and fragments thereof of 15 ormore nucleotides.

In yet another embodiment, the present invention includes a set ofbi-directional primary sequencing primers includes a second primercomprising the sequence of SEQ ID NO:30.

In a yet another aspect of the present invention, the primers used tosequence the HR1 domain of HIV-1 will have a sequence selected from thegroup consisting of one or more of SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36 (set forth below) orfragments thereof of at least 15 or more nucleotides.

SEQ ID NO:31 5′-TTGGGTTCTTGGGAGCAGCAGGAAG-3′ SEQ ID NO:325′-TTGGGTTCTTGGGAGCAGCAGG-3′ SEQ ID NO:335′-AGTRGTGCARATGAKTTTTCCAGAG-3′ SEQ ID NO:345′-GTGGTGCAGATGAGTTTTCCAGAG-3′ SEQ ID NO:355′-GTGGTGCAGATGAGTTTTCCAGAGC-3′

SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33 represent alternate forwardprimer sequences in the region to the 5′ side of HR1. The primersequence of SEQ ID NO:31 corresponds to the sequence of nucleotides7329-7353 of SEQ ID NO:1. The primer sequence of SEQ ID NO:32corresponds to the sequence of nucleotides 7329-7350 of SEQ ID NO:1. Theprimer sequence of SEQ ID NO:33 corresponds to the sequence ofnucleotides 7337-7361 of SEQ ID NO:1.

SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 represent alternate reverseprimer sequences in the region to the 3′ side of HR1 (between the HR1and HR2 domains). The primer sequence of SEQ ID NO:34 corresponds to thereverse complement of nucleotides 7563-7587 of SEQ ID NO:1(ctctggaaaamtcatytgcacyact). The primer sequence of SEQ ID NO:35corresponds to the reverse complement of nucleotides 7564-7587 of SEQ IDNO:1 (ctctggaaaactcatctgcaccac). The primer sequence of SEQ ID NO:36corresponds to the reverse complement of nucleotides 7563-7588 of SEQ IDNO:1 (ctctggaaaactcatctgcaccacg).

In one embodiment of the present invention, the primer includes thenucleotide sequence of SEQ ID NO:1 and fragments thereof of 15 or morenucleotides.

In another embodiment of the present invention, the primer includes thenucleotide sequence of SEQ ID NO:31 and fragments thereof of 15 or morenucleotides.

In another embodiment of the present invention, the primer includes thenucleotide sequence of SEQ ID NO:32 and fragments thereof of 15 or morenucleotides.

In another embodiment of the present invention, the primer includes thenucleotide sequence of SEQ ID NO:33 and fragments thereof of 15 or morenucleotides.

In another embodiment of the present invention, the primer includes thenucleotide sequence of SEQ ID NO:34 and fragments thereof of 15 or morenucleotides.

In another embodiment of the present invention, the primer includes thenucleotide sequence of SEQ ID NO:35 and fragments thereof of 15 or morenucleotides.

In another embodiment of the present invention, the primers includes aset of bidirectional sequencing primers, wherein the forward primer isselected from the group consisting of SEQ ID NO:1, SEQ ID NO:31, and SEQID NO:32, and fragments thereof of 15 or more nucleotides, and thereverse primer is selected from the group consisting of SEQ ID NO:33,SEQ ID NO:34, and SEQ ID NO:35, and fragments thereof of 15 or morenucleotides.

The forward primer site, illustrated by SEQ ID NO:1, SEQ ID NO:31, andSEQ ID NO:32, the 3′ terminal end is preferably a -G nucleotide base,and more preferably a -GG. This allows particularly strong hybridizationat the 3′ end of the primer and possibly enhanced sequencing resultsversus a primer that ends at a different location. The 3′ end of theprimer may extend further in the 3′ direction. Due to the fact thatsequence immediately following primers is generally of poor quality, the3′ end of the forward primer will preferably be located a sufficientdistance from the HR1 region that the beginning of the HR1 sequence isunequivocal. Preferably, the 3′ end of the forward primer will extend nofurther than the -AAG at the 3′ end of SEQ ID NO:1, although primersextending beyond the point are contemplated, provided suitable sequencefor the HR1 region can be obtained.

The 5′ end of this primer could be shortened slightly, but would alsochange the annealing characteristics of the primer. Specifically, the 5′terminal -GG may be shortened to -G. The shorter versions (TGG-, GG-,G-) could be combined with the extension of the 3′ end to furtheroptimize the annealing (or melting temp, Tm) temperature to match otherpossible reverse primers.

The forward primers may be modified beyond the locations described abovewith appropriate modifications to the sequencing chemistry used. Forinstance, universal sequencing primer tails could be utilized on anested PCR primer to move the primer inward a bit without putting theuniversal sequencing primer too close to the critical region. This wouldrequire that the WP primer include a universal sequencing tail as a PCRprimer.

The reverse primers, illustrated by SEQ ID NO:33, SEQ ID NO:34, and SEQID NO:35, may also be modified in accordance with the present invention.In preferred embodiments, the 5′ end of the reverse primer may benucleotide 7564 (G) or 7563 (A). In preferred embodiments, the 3′ end ofthe reverse primer may be nucleotide 7585 (G), 7586 (A), 7587 (G) or7588 (C). The primer will preferably not be located any further from theHR1 region, which would place the sequence in less conserved HIVsequence positions and result in a less robust sequencing primer set.The primer will preferably not be located any closer to the HR1 region,which may not provide sufficient initial sequence immediately followingthe primer to allow the sequence data to be resolved before the HR1positions are reached.

The primer combinations described above can be used in a method inaccordance with the invention for a sample suspected of containing theHIV-1 virus to assess the subtype and genotype of the virus.

The method comprises the steps of treating the sample to recover viralRNA; reverse transcribing the recovered viral RNA; sequencing thereverse transcription product; and using the results of the sequencingstep to establish the genotype of the tested virus. In this method,either or both of the reverse transcription step and the sequencing stepare performed using primer combinations as described above. The methodof the invention can include the step of performing a parallelgenotyping procedure that is designed to evaluate polymorphic variationsin the gp41 region of HIV, and particularly for evaluating polymorphicvariations in the HR1 domain of gp41. Alternatively, the method can beutilized with a sample that has previously been the subject of a failedgenotyping attempt using genotyping procedures specific for gp41.

Kits

The present invention also includes kits comprising reagents necessaryand sufficient to perform the methods described above. In one aspect,the present invention is directed to a kit for detecting the presence orabsence of a mutation of interest in a pathogen in a sample containingmultiple quasispecies of the pathogen having mixed length polymorphisms,wherein the mutation of interest is located adjacent to the lengthpolymorphism, comprising a first primer for sequencing a first strand ofa region of a DNA template containing the mutation of interest and asecond primer for sequencing a second strand of a region of the DNAtemplate containing the mutation of interest, wherein the region definedby (i.e., including and between) the first primer and second primerexcludes the length polymorphism.

In another aspect, the present invention is directed to a kit comprisingone or more an oligonucleotide primers selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and fragmentsthereof of 15 or more nucleotides.

In yet another aspect, the present invention is directed to a kitcomprising:

-   -   (a) a forward primer selected from the group consisting of one        or more of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,        SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID        NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,        and fragments thereof of 15 or more nucleotides; and    -   (b) a reverse primer selected from the group consisting of one        or more of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID        NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,        SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID        NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and fragments        thereof of 15 or more nucleotides.

In yet another embodiment, the present invention is directed to a kitcomprising an oligonucleotide primer capable of generating primerextension products corresponding to the region consisting essentially ofnucleotides 7329-7614 of SEQ ID NO:1, or fragments thereof of 15 or morenucleotides.

In yet another embodiment, the present invention is directed to a kitcomprising an oligonucleotide primer, wherein the primer is capable ofgenerating primer extension products corresponding to the regionconsisting essentially of nucleotides 7388-7549 of SEQ ID NO:1 orfragments thereof of 15 or more nucleotides.

In yet another embodiment, the present invention is directed to a kitcomprising an oligonucleotide primer, wherein the primer is capable ofgenerating primer extension products corresponding to the regionconsisting essentially of nucleotides 7492-7522 of SEQ ID NO:1 orfragments thereof of 15 or more nucleotides.

In yet another embodiment, the present invention is directed to a kitcomprising an oligonucleotide primer, wherein the primer is selectedfrom the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,SEQ ID NO:34, SEQ ID NO:35, and fragments thereof of 15 or morenucleotides.

In yet another embodiment, the present invention is directed to a kitcomprising a primer combination comprising a set of bi-directionalsequencing primers encompassing a region consisting essentially ofnucleotides 7329-7614 of SEQ ID NO:1 or fragments thereof of 15 or morenucleotides.

In yet another embodiment, the present invention is directed to a kitcomprising a primer combination, wherein the set of bi-directionalsequencing primers encompasses a region consisting essentially ofnucleotides 7388-7549 of SEQ ID NO:1 or fragments thereof of 15 or morenucleotides.

In yet another embodiment, the present invention is directed to a kitcomprising a primer combination, wherein the set of bi-directionalsequencing primers encompasses a region consisting essentially ofnucleotides 7492-7522 of SEQ ID NO:1 or fragments thereof of 15 or morenucleotides.

In yet another embodiment, the present invention is directed to a kitcomprising a primer combination, wherein the set of bi-directionalprimary sequencing primers comprises two or more primers selected fromthe group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, and fragments thereof of 15 or more nucleotides.

In yet another embodiment, the present invention is directed to a kitcomprising an primer combination, wherein the set of bi-directionalsequencing primers comprises:

-   -   (a) at least one forward primer selected from the group        consisting of SEQ ID NO:31 and SEQ ID NO:32, and fragments        thereof of 15 or more nucleotides, and    -   (b) at least one reverse primer selected from the group        consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and        fragments thereof of 15 or more nucleotides.

The following example illustrates particular embodiments of the presentinvention.

EXAMPLE 1

The following example illustrates bi-directional (dual strand)sequencing with a primary sequencing primer set (EC7719F, EC8311R) and asecondary sequencing primer set (WP8746F, WP8960RB and/or WP8746RC) inthe HR1 region of gp41 from a clinical plasma sample exhibiting a mixedpopulation, including a length polymorphism. A complex sequence traceobtained using only the forward strand primary sequencing primer of anearly equal mixture with and without the length polymorphism showedinconclusive results. Routine analysis does not permit deconvolution ofthe resulting superimposed peaks. As demonstrated below, use of thesecondary primer set produces immediately usable bi-directional results.

A representative set of combined RT-PCR and CLIP sequencing conditionswas used to generate high quality sequence data using the primers:

EMF1 (5′-AGAGAAAGAGCAGAAGACAGTGGC-3′), and EMR1(5′-CCTTGTAAGTCATTGGTCTTAAAGGTACC-3′) (RT-PCR primers); EC7719F(5′-ACCAAGGCAAAGAGAAGAGTGG-3′), and EC8311R(3′-gtaggaggcttgataggtttaag-5′) (CLIP primers); and WP8746F(5′-TTGGGTTCTTGGGAGCAGCAGG-3′), WP8960RB(5′-GTGGTGCAGATGAGTTTTCCAGAG-3′) and/or WP8746RC(5′-GTGGTGCAGATGAGTTTTCCAGAGC-3′) (CLIP primers).

The following materials were used:

RT-PCR Reagents: TRUGENE HIV Kit CLIP Reagents: VG 30001 Core SequencingKit RT-PCR Primers: EMF1 (unlabelled) 30 μM EMR1 (unlabelled) 30 μM CLIPPrimers: EC7719F (Cy 5.5) 3 μM EC8311R (Cy 5.0) 3 μM EC8311R(unlabelled) 3 μM

RNA was extracted from patient plasma samples according to the packageinstructions in a TRUPREP Extraction Kit for viral RNA.

For RT-PCR amplification of the gp41 region, the following reagents wereused:

Primer stock volume (μL) required Number of samples conc. (μM) persample Master Mix I RT-PCR Primers Forward (μL)* 30 0.35 RT-PCR PrimersReverse (μL)* 30 0.35 Nuclease free H2O (μL)** 14.47 dNTP Solution (μL)1.75 DTT Solution (μL) 1.16 RNase-Inhibitor (μL) 0.59 TOTAL (μL)** 18.67Master Mix II RT-PCR Buffer (μL) 11.67 RNase-Inhibitor (μL) 0.58 RTEnzyme (μL) 1.17 DNA Polymerase (μL) 2.92 TOTAL (μL) 16.34

Master Mix I and II were maintained on ice until use. 16 μL of MasterMix I was aliquoted to the bottom of each PCR tube. 10 μL each of theextracted RNA sample was the pipetted into the PCR plate containingMaster Mix I. The tubes were then placed on the thermocycler. After 5minutes of the single 50° C. cycle, the thermocycler was paused and 14μL of Master Mix II was added to each well. The thermocycler program wasthen resumed according to the following protocol:

 1x 90° C. for 2 minute 50° C. for 20 minute Note: pause after 5 minutesof 50° C. step above to add Master Mix II 94° C. for 2 minute 37x 94° C.for 30 seconds 60° C. for 30 seconds 68° C. for 2 minute  1x 68° C. for7 minute  4° C. hold until operator action

The resulting PCR amplification product was then sequenced as follows,using the CLIP sequencing method. First, the 7.00 μL Thermo Sequenaseenzyme was diluted 10-fold with 63 μL enzyme dilution buffer, to a totalvolume of 70.00 μL. 11.50 μL of labeled EC8311R (3 μL) reverse primerwas then diluted 50% with 11.50 μL of unlabeled EC8311R (3 μL) reverseprimer, to a total volume of 23.00 μL. The gp41 CLIP Master Mix was thenprepared as follows:

volume (μL) required gp41 CLIP Master Mix Master Mix per sampleSequencing Buffer (μL) 2.75 EC7719F (3 uM) (μL) 1.38 EC8311R primer mix1.38 DNA (μL) 0.00 DMSO (μL) 0.00 dH2O (μL) 8.80 1:10 diluted ThermoSequenase (μL) 4.40 Total (μL) 18.71

17 μL of CLIP Master Mix was added to 5 μL of each amplification productto be sequenced. 3 μL of the termination mix A, C, G, T was thenpipetted into each sequencing tube

5 μL of the Master Mix/amplification product was pipetted into eachsequencing tube containing the termination mixes. The mixes were thensequenced using the following CLIP Sequencing Thermocycling program:

1x 94° C. for 5 minute (30)x 94° C. for 20 seconds 60° C. for 20 seconds70° C. for 1.5 minute 1x 70° C. for 5 minute  4° C. hold

Once sequencing was complete, 6 μL stop loading dye was added into eachtube. Samples were heated at 94° C. for 2 min, quenched on ice and mixedwell by gentle vortexing. 2 μL were loaded into each well of theMicrocel™ cassette. The TRUGENE protocol for LRTower setup and loading,as provided by the manufacturer, was then followed for automated DNAsequencing, using Microcel 500 and 6% Surefill, with 2000V, 50% laserpower and 70 minute run time.

Table 1 and Table 2 below show analysis of results of sequencing the HR1and HR2 regions combined (Table 1) and sequencing the HR1 region alone(Table 2). As shown in Table 1, the “ambiguous matches” column matchesat least one base or code with an ambiguous base-call at the position ineach of the FASTA files.

TABLE 1 HR1 and HR2 Regions Combined Strictly Ambiguous All Mis- Mis-Alignment % Sample Mismatches matches matched Length Consistency22871.msf 26 26 0 486 100.0 28942.msf 22 23 1 485 99.8 35794.msf 30 32 2487 99.6 37047.msf 31 34 3 487 99.4 61919.msf 7 7 0 486 99.4 62290.msf22 23 1 494 99.8 62291.msf 15 15 0 488 100.0 62292.msf 11 11 0 486 100.062293.msf 17 17 0 486 100.0 62294.msf 9 10 1 487 99.8 62295.msf 43 43 0486 100.0 62316.msf 14 14 0 489 100.0 62317.msf 11 13 2 485 99.662423.msf 10 10 0 486 100.0 62424.msf 10 11 1 486 99.8 63009.msf 10 12 2486 99.6 63680.msf 88 94 6 492 98.8 pc.msf 7 9 2 486 99.6

The above data demonstrates that primers encompassing the HR1 and HR2regions of HIV-1 are able to obtain sequence data for a broad range ofHIV variants.

Table 2 demonstrates that in preferred aspects of the invention, inwhich the primers encompass the HR1 region alone, fewer ambiguousmatches in the HR1 region result in fewer editing steps and higheroverall consistency.

TABLE 2 HR1 Region Only Strictly Ambiguous All Mis- Mis- Alignment %Sample Mismatches matches matched Length Consistency 22871.msf 2 2 0 273100.0 28942.msf 9 9 2 272 100.0 35794.msf 19 21 1 273 99.3 37047.msf 1920 0 273 99.6 61919.msf 3 3 0 273 100.0 62290.msf 5 5 0 273 100.062291.msf 0 0 0 273 100.0 62292.msf 2 2 0 273 100.0 62293.msf 2 2 0 273100.0 62294.msf 4 4 0 274 100.0 62295.msf 27 27 0 273 100.0 62316.msf 12 0 273 100.0 62317.msf 5 7 2 272 99.3 62423.msf 3 3 0 273 100.062424.msf 0 0 0 273 100.0 63009.msf 3 3 0 273 100.0 63680.msf 45 45 0273 100.0 pc.msf 0 0 0 273 100.0

With reference to a specific patient sample, sequence traces from sample28942 were generated using both the EC primer set and the WP primer set.The sequence data generated with only the EC primer set resulted insequence data that was readable and could be confirmed in unreadablefrom one direction, resulting in ambiguous results that could not beconfirmed. When sample 28942 was analyzed with the WP primer set,however, the ambiguous data was confirmed with the antisense strandsequence.

The above data demonstrates that sequencing a region consistingessentially of the HR1 domain, using the primers of the presentinvention, result in significantly fewer ambiguities and mismatches, andimproved accuracy and consistency of sequencing results. For example,the following examples of HIV-1 gp41 sequencing data generated in thearea of particular interest for enfuvirtide (FUZEON™ or T-20) treatmentassociated resistance mutations (HR1) include data with the standarddesign primers (EC) and the new primers (WP). In general any mixedlength polymorphism mutation that occurs between one of the outerprimers and the mutations of interest causes the sequence quality todecline due to overlayed and out of frame sequences being generated. Theprimers of the present invention circumvent the problem and produce highquality bidirectional sequence data in the area of interest, allowingantisense strand confirmation of any mutations that occur in HR1.

1. A primer combination comprising a set of bi-directional sequencingprimers encompassing a region comprising the HR1 and HR2 domains ofHIV-1, wherein the primer combination comprises: (a) a forward primerselected from the group consisting of one or more of SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO: 13, SEQID NO:14, and fragments thereof of 15 or more nucleotides; and (b) areverse primer selected from the group consisting of one or more of SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, and fragments thereof of 15 or more nucleotides.
 2. A primercombination according to claim 1, wherein the primer combinationcomprises a set of bi-directional sequencing primers encompassing aregion consisting essentially of nucleotides 7329-7614 of SEQ ID NO:1 orfragments thereof of 15 or more nucleotides.
 3. The primer combinationaccording to claim 2, wherein the set of bi-directional sequencingprimers encompasses a region consisting essentially of nucleotides7388-7549 of SEQ ID NO:1 or fragments thereof of 15 or more nucleotides.4. The primer combination according to claim 3, wherein the set ofbi-directional sequencing primers encompasses a region consistingessentially of nucleotides 7492-7522 of SEQ ID NO:1 or fragments thereofof 15 or more nucleotides.
 5. The primer combination according to claim4, wherein the set of bi-directional primary sequencing primerscomprises two or more primers selected from the group consisting of SEQID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, andfragments thereof of 15 or more nucleotides.
 6. The primer combinationof claim 5, wherein the set of bi-directional sequencing primerscomprises: (a) at least one forward primer selected from the groupconsisting of SEQ ID NO:31 and SEQ ID NO:32, and fragments thereof of 15or more nucleotides, and (b) at least one reverse primer selected fromthe group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, andfragments thereof of 15 or more nucleotides.
 7. A method for detectingthe presence or absence of a mutation of interest in the nucleic acid ofa pathogen, wherein the mutation of interest is located adjacent to alength polymorphism defining multiple quasispecies of the pathogen,comprising: a) obtaining from the patient sample a double-stranded DNAtemplate encompassing the mutation of interest; b) sequencing a firststrand of a region of the DNA template containing the mutation ofinterest; c) sequencing a second strand of a region of the DNA templatecontaining the mutation of interest, wherein the region of the DNAtemplate sequenced that is common to the first strand and second strandexcludes the length polymorphism; and d) comparing the sequence of thefirst strand with the sequence of the second strand to obtaincomplementary strand confirmation of the sequence of the mutation ofinterest.
 8. A method according to claim 7, wherein the pathogen isHIV-1.
 9. The method according to claim 8, wherein the mutation ofinterest is located in the HIV-1 envelope glycoprotein gp41.
 10. Themethod of claim 9, wherein the mutation of interest is located withinposition 36-45 of gp41.
 11. The method according to claim 7, wherein theregion of the DNA template sequenced that is common to the first strandand second strand and excludes the length polymorphism consistingessentially of nucleotides 7329-7614 of SEQ ID NO:1 or fragments thereofof 15 or more nucleotides.
 12. The method according to claim 7, whereinthe region of the DNA template sequenced that is common to the firststrand and second strand and excludes the length polymorphism consistingessentially of nucleotides 7388-7549 of SEQ ID NO:1 or fragments thereofof 15 or more nucleotides.
 13. The method according to claim 7, whereinthe region of the DNA template sequenced that is common to the firststrand and second strand and excludes the length polymorphism consistingessentially of nucleotides 7492-7522 of SEQ ID NO:1 or fragments thereofof 15 or more nucleotides.
 14. The method according to claim 7, whereinthe sequence is determined using primers selected from the groupconsisting of one or more primers selected from the group consisting ofSEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,and fragments thereof of 15 or more nucleotides.
 15. The methodaccording to claim 7, wherein the sequence is determined using forwardprimers comprising one or more primers selected from the groupconsisting of SEQ ID NO:31, SEQ ID NO:32, and fragments thereof of 15 ormore nucleotides, and reverse primers comprising one or more primersselected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, and fragments thereof of 15 or more nucleotides.
 16. The methodaccording to claim 7, wherein the sequence is determined using primersselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, and fragments thereof of 15 or more nucleotides.
 17. The methodaccording to claim 16, wherein the sequence is determined using aforward primer selected from the group consisting of one or more of SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, and fragments thereof of 15 or morenucleotides.
 18. The method according to claim 16, wherein the primer isa reverse primer selected from the group consisting of one or more ofSEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, and fragments thereof of 15 or more nucleotides.
 19. Themethod according to claim 16, comprising a primer combination comprisinga set of bi-directional sequencing primers encompassing a regionencompassing the HR1 and HR2 domains of HIV-1, wherein the primercombination comprises: (a) a forward primer selected from the groupconsisting of one or more of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, andfragments thereof of 15 or more nucleotides; and (b) a reverse primerselected from the group consisting of one or more of SEQ ID NO:15, SEQID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, andfragments thereof of 15 or more nucleotides.
 20. A kit for detecting thepresence or absence of a mutation of interest in a pathogen in a samplecontaining multiple quasispecies of the pathogen having mixed lengthpolymorphisms, wherein the mutation of interest is located adjacent tothe length polymorphism, comprising a first primer for sequencing afirst strand of a region of a DNA template containing the mutation ofinterest and a second primer for sequencing a second strand of a regionof the DNA template containing the mutation of interest, wherein theregion defined by the first primer and second primer excludes the lengthpolymorphism.
 21. A kit according to claim 20, wherein the kit comprisesone or more an oligonucleotide primers selected from the groupconsisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,SEQ ID NO:35, and fragments thereof of 15 or more nucleotides.
 22. A kitaccording to claim 21, wherein the kit comprises one or more anoligonucleotide primers selected from the group consisting of SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, and fragments thereof of 15 or morenucleotides.